Purified nitric oxide synthase

ABSTRACT

The present invention provides a novel constitutive nitric oxide synthase (NOS) that utilizes both L-arginine and arginine-rich peptides, oligopeptides or proteins, e.g., bradykinin (BK), as substrates in the synthesis of nitric oxide (NO). Also provided are methods of controlling, regulating or modulating NO synthesis in a subject.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of copendingapplication Ser. No. 08/675,821, filed Jul. 5, 1996, the disclosure ofwhich is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention is directed to a purified enzyme and, moreparticularly, to a novel, purified, constitutive mammalian nitric oxidesynthase (NOS) that utilizes both L-arginine and arginine-rich peptides,oligopeptides (e.g., bradykinin (BK)), and proteins as substrates.

[0004] 2. Description of Related Art

[0005] Nitric oxide synthases (NOS, EC 1.14.23) are important enzymes,which convert L-arginine to L-citrulline and nitric oxide (NO). Nitricoxide is a very short-lived free radical, which is rapidly oxidized tonitrite (NO₂) and nitrate (NO₃) which are measured as the stableinactive end products of nitric oxide formation. The significance,however, lies in the fact that NO appears to play a pivotal role in awide variety of physiological and pathological processes in mammals.These processes include vasodilation and regulation of normal vasculartone, inhibition of platelet aggregation, neuronal transmission,cytostasis, hypotension associated with endotoxic shock, inflammatoryresponse-induced tissue injury, mutagenesis, and formation ofcarcinogenic N-nitrosamines (Nathan, FASEB J. 6:3051-3064 (1992);Kiechle et al., Am. J. Clin. Pathol. 100:567-575 (1993)). For example,it is well-known in the art to treat humans afflicted with anginadistress and cardiovascular disease with nitroglycerin, which acts as avasodilating agent. In the body, nitroglycerin is converted to nitricoxide (NO), which is the pharmacologically active metabolite. See,Palmer et al., Nature 333:664-666 (1988). Thus, evidence that NOmediates functions as diverse as those which occur in the brain, theendothelium and the blood, has led to intense study into the biologicalroles of NO and the various distinct members of the NOS family. (See,e.g., Marletta, J. Biol. Chem. 268:12231-12234 (1993); Knowles et al.,Biochem. J. 298:249-258 (1994)). It is well known by those skilled inthe art that multiple isoforms of the NOS enzyme exist and that they aregenerally classified into two broad categories: 1) constitutive and 2)inducible. These classes of NOS enzymes vary considerably in their size,amino acid sequence, activity and regulation, and exhibit a number ofsubstantial differences, indicating differences in their molecularstructures. Increasingly diverse biological functions are beingattributed to the NO formed by these three major known types of NOS(Nathan, FASEB J. 6:3051 (1992); Marletta, J. Biol. Chem. 268:12231(1993); Knowles et al., Biochem. J. 298:249 (1994); Griffith et al.,Annu. Rev. Physiol. 57:707 (1995)). For example, cells such as neuronsand vascular endothelial cells contain constitutive NOS isotypes, whilemacrophages and vascular endothelial cells express an inducible NOS.

[0006] Several isoforms of NOS's from different mammalian tissues andcells have been purified and characterized; in brain (nNOS) by Bredt andSynder, Proc. Natl. Acad. Sci. USA 87:682-685 (1990); in endothelialcells (eNOS) by Forstermann et al., Biochem. Pharmacol. 42:1849-1857(1991); in macrophages (iNOS) by Hibbs et al., Science 235:473 (1987)and Stuehr et al., Proc. Natl. Acad. Sci U. S. A. 88:7773-7777 (1991);in hepatocytes by Knowles et al., Biochem. J. 279:833-836 (1990); invascular cells by Wood et al., Biochem. Biophys. Res. Comm. 170:80-88;in neutrophils by Yui et al., J. Biol. Chem. 266:12544-12547 (1991) andYui et al., J. Biol. Chem. 266:3369-3371 (1991); and in other tissues(see, e.g., Hevel et al., J. Biol. Chem. 266:22789-22791 (1991); Mayeret al., FEBS Lett. 277:215-219 (1990); Schmidt et al., Proc. Natl. Acad.Sci U S. A. 88:365-369 (1991); Ohshima et al., Biochem. Biophys. Res.Commun. 183:238-244 (1992); Hiki et al., J. Biochem. 111:556-558 (1992);Evans et al., Proc. Natl. Acad. Sci U. S. A. 89:5361-5365 (1992);Sherman et al., Biochemistry 32:11600-11605 (1993)). U.S. Pat. No.5,268,465 claims a cDNA molecule encoding all or a portion of amammalian calmodulin-dependent nitric oxide synthase (nNOS), comprisingbetween 12 and 4,000 nucleotides. U.S. Pat. No. 5,468,630 claims anisolated nucleic acid molecule comprising the nucleic acid sequenceencoding a human inducible nitric oxide synthase (iNOS) protein. U.S.Pat. No. 5,498,539 claims an isolated nucleic acid molecule comprisingthe nucleic acid sequence encoding a bovine endothelial nitric oxidesynthase (eNOS) protein having amino acid or nucleic acid sequences setforth in the specification.

[0007] It is also known that small amounts of NO generated by aconstitutive NOS appear to act as a messenger molecule by activatingsoluble guanylate cyclase and, thus, increasing intracellular guanosine,3′, 5′-cyclic monophosphate (cGMP) and the induction of biologicalresponses that are dependent on cGMP as a secondary messenger. Forexample, through this mechanism, endothelial derived NO inducesrelaxation of vascular smooth muscle and is identified as endotheliumderived relaxing factor (Palmer et al., Nature 327:524-526 (1987) andIgnarro et al., Proc. Natl. Acad. Sci. USA 84:9265-9269 (1987)). Inaddition, neuronal nitric oxide can act as a neuro-transmitter byactivating guanylate cyclase with important functions in the centralnervous system and autonomic nervous systems. Bredt and Synder, Proc.Natl. Acad. Sci. USA 86:9030-9033 (1989) and Burnett et al., Science257:401-403 (1992). Moreover, various purified NOS enzymes have beenidentified as hemeproteins (Stuehr et al., J. Biol. Chem.267:20547-20550 (1992); White et al., Biochemistry 31:6627-6631 (1992);McMillan et al., Proc. Natl. Acad. Sci. USA 89:11141-11145 (1992)) andflavoproteins (Hevel et al., J. Biol. Chem. 266:22789-22791 (1991);Bredt et al., J. Biol. Chem. 267:10976-10981 (1992)).

[0008] Thus, the catalytic mechanisms of the NOS enzymes have also beenthe subject of great interest (see, e.g., Marletta, J. Biol. Chem.268:12231-12234 (1993)). Stable isotope studies have shown when thatwhen L-arginine is the substrate for the enzyme, NO derives from one ofthe two equivalent guanidino nitrogens on the arginine moiety (Ignarroet al., 1987; Palmer et al., 1988), and that di-oxygen is the source ofthe oxygen atoms incorporated into citrulline and NO (Kwon et al., J.Biol. Chem. 265:13442-13445 (1990); Leone et al., J. Biol. Chem.266:23790-23795 (1991)). Moreover, N^(G)-hydroxy-L-arginine has beendemonstrated to be an oxidative intermediate in the catalytic process(Stuehr et al., J. Biol. Chem. 266:6259-6263 (1991); Wallace et al.,Biochem. Biophys. Res. Commun. 176:528-534 (1991); Pufahl et al.,Biochemistry 31:6822-6828 (1992); Klatt et al., J. Biol. Chem.268:14781-14787 (1993)).

[0009] Co-factors involved in the conversion of L-arginine toL-citrulline and NO synthesis include tetrahydrobiopterin (BH₄), flavinadenine nucleotide (FAD) , the flavin-ribitol phosphate part of FAD(FMN) and the reduced form of nicotinamide adenine dinucleotidephosphate (NADPH). In addition, when the NOS enzyme has been derivedfrom brain or endothelial cells, calcium and calmodulin are alsorequired. (Bredt et al., Nature 351:714-718 (1991); Lamas et al., Proc.Natl. Acad. Sci. U. S. A. 89:6348-6352 (1992); Lyons et al., J. Biol.Chem. 267:6370-6374 (1992); Lowenstein et al., Proc. Natl. Acad. Sci. U.S. A. 89:6711-6715 (1992); Xie et al., Science 256:225-228 (1992)).Calmodulin is a well-known protein binder for Ca²+, ubiquitously foundin plant and animal cells. The Ca²⁺-calmodulin complex thus formed isknown to bind to various target proteins in the cell, and thereby altertheir activity.

[0010] Thus, it will be appreciated that nitric oxide has both normalphysiologic intracellular and extracellular regulatory functions.However, excessive production of nitric oxide is detrimental. Forexample, when vascular endothelial cells are stimulated to express a NOSenzyme by a bacterial endotoxin, such as for example bacteriallipopolysaccharide (LPS), and inflammatory cytokines are elevated, theexcess amounts of nitric oxides that are produced contribute to thevascular collapse seen in sepsis. Busse and Mulsch, FEBS Lett.265:133-136 (1990). It is also known that when vascular cells arestimulated to express a NOS enzyme by inflammatory cytokines, the excessamounts of nitric oxides cause massive dilation of blood vessels andsustained hypotension commonly encountered in septic shock, andcontribute to the eventual vascular collapse seen in sepsis. Id. It isalso known that overproduction of nitric oxide in the lungs stimulatedby immune complexes directly damages the lung. Mulligan et al., J.Immunol. 148:3086-3092 (1992). Induction of nitric oxide synthase inpancreatic islets impairs insulin secretion and contributes to the onsetof juvenile diabetes. Corbett et al., J. Biol. Chem. 266:21351 (1991).

[0011] Thus, it will be appreciated that there is a great need in themedical community for the ability to control and regulate specific formsof NOS, particularly given its role in maintaining normal blood pressureand the devastating effect of excess NO on the cardiovascular,gastrointestinal and respiratory systems in humans. Thus considerableresearch has been expended to discover inhibitors and regulators of NOSactivity. However, until the present invention, researchers have limitedtheir work almost exclusively to the premise that L-arginine is the mostphysiologically relevant substrate of NOS. The studies have extended nofurther than the fact that dipeptides, such as Arg-Arg and Arg-Phe arealso oxidized by crude NOS preparations derived from culturedendothelial cells and macrophages (Hecker et al., FEBS Lett. 294:221(1991); Hecker et al., Proc. Natl. Acad. Sci. USA 87:8612 (1990); Heckeret al., J. Cardiovasc. Pharmacol. 20:S139 (1992)). Thus, although therehave been many publications directed to analyses of arginine analogs orderivatives which inhibit NOS activity by blocking the use of arginineas a substrate for NO synthesis, or to the cofactors necessary for theconversion of arginine, no one until the present inventors hasconsidered peptide, oligopeptide or protein substrates for the NOSenzyme. Hence, given the expectation that natural or syntheticarginine-rich peptide, oligopeptide or protein antagonists can functionas NOS inhibitors, the present invention will greatly expand the numberand types of inhibitors available for the regulation and control of NOproduction in the body. In addition, the use of a NOS to supplydeficient individuals with NO-forming capability will be greatlyenhanced by the purification of the nNOS-II class of enzymes. Thepresent invention, therefore, will also provide many new ways to studythe biological mechanisms involved in NO synthesis.

SUMMARY OF THE INVENTION

[0012] The present invention relates to a novel constitutive nitricoxide synthase, referred to hereinafter as nNOS-II, that has beenpurified from mammalian neural tissue, and which is capable of utilizingarginine-rich peptides, polypeptides and proteins (e.g., bradykinin), aswell as L-arginine, as substrates. Unlike previously defined NOSisoforms, nNOS-II is unique in that it is calmodulin-dependent withL-arginine as substrate, but calmodulin-independent with anarginine-rich polypeptide, such as bradykinin (BK), as substrate. WhenBK is the substrate, both the N- and the C-terminal arginines of theoligopeptide are oxidized to citrullines by nNOS-II. See Equation I.

[0013] Moreover, with BK as substrate, NOS activity is competitivelyinhibited by N^(G)-methyl-, N^(G)-nitro-L-arginines, and BKreceptor-antagonists, including oligopeptide BK receptor-antagonists.

[0014] More particularly, the present invention relates to asubstantially purified nNOS-II protein.

[0015] In another embodiment, the present invention relates to amammalian brain-derived nitric oxide synthase protein purified to anactivity at least 6,360-fold, said protein having a denatured molecularmass as determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis under reducing conditions of about 105 kD, and a nativehomodimeric molecular mass as determined by gel filtration of about 230kD, requiring NADPH, FAD, FMN, Ca²⁺ and tetrahydrobiopterin cofactorsfor the production of nitric oxide either from L-arginine, or an analogor derivative thereof, or from an arginine-rich peptide, oligopeptide,or protein substrate. As used herein, the term “peptide” is arbitrarilydefined as a peptide chain having a single peptide bond, “oligopeptide”is a peptide chain having from 2 to 14 peptide bonds, inclusive, and“protein” is a peptide chain having 15 or more peptide bonds.“Polypeptide” is intended to be generic to “oligopeptide” and “protein.”“Arginine-rich” means that the peptide, oligopeptide, or protein has atleast one sterically accessible arginine moiety.

[0016] In another embodiment, the present invention relates to a methodof regulating or controlling nitric oxide production in a mammaliansubject comprising administering to the mammal a nitric oxide-regulatingamount of a peptide, oligopeptide, or protein inhibitor of nitric oxidesynthase, preferably nNOS-II. As used herein, “mammalian subject” isintended to include human subjects.

[0017] In one embodiment, the present invention relates to a method ofreducing the rate of nitric oxide production in a mammalian subjectcomprising administering to the mammal a nitric oxide inhibiting amountof a peptide, oligopeptide or protein inhibitor of nitric oxidesynthase, preferably nNOS-II. “Reducing the rate” is intended to includea reduction to zero, in which case the reduction would be understood toinclude, not only a decrease in the rate of NO production or of thequantity of NO produced, but also a prevention of excess NO production.

[0018] In another embodiment, the present invention relates to a methodof enhancing the rate of nitric oxide production in a mammalian subjectcomprising administering to the mammal a nitric oxide enhancing amountof nitric oxide synthase, preferably nNOS-II. “Enhancing the rate” isintended to include a stimulating, inducing or causing production of NOin a NO-deficient or NO-defective subject, in which case the enhancementwould be understood to include stimulation, induction or initializationof NO production in such subjects or individuals, as well as increasingthe rate of NO production or the quantity of NO produced.

[0019] In still another embodiment, the present invention relates to amethod of preventing or treating a nitric oxide-mediated disease orcondition in a mammalian subject comprising administering to the subjectin need of such prevention or treatment a therapeutically effectiveamount of a peptide, oligopeptide, or protein inhibitor of nitric oxidesynthase, preferably nNOS-II.

[0020] Additional objects, advantages and novel features of theinvention will be set forth in part in the description which follows,and in part will become apparent to those skilled in the art onexamination of the following, or may be learned by practice of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 depicts a DEAE-agarose column elution profile for both NOSenzymes from rat cerebellum. L-arginine (◯) NOS activity (calmodulindependent), and BK () NOS activity (calmodulin independent). Proteinabsorbance at 280 nm (□) from rat cerebellum was used to monitor theeluting process.

[0022]FIG. 2 depicts an 8%, silver-stained SDS-polyacrylamide gelshowing: Lane 1, cytosol (10 μg protein); Lane 2, eluate from firstDEAE-column (10 μg); Lane 3, NADPH eluate from 2′,5′-ADP-agarose column(2 μg protein); Lane 4, eluate from second DEAE-agarose columncontaining purified enzyme (0.5 μg protein); Lane 5, control with samplebuffer; Lane 6, molecular weight markers (Bio-Rad) of myosin (208,000),b-galactosidase (115,000) phosphorylase B (107,000), bovine serumalbumin (79,500), ovalbumin (49,500), carbonic anhydrase (34,800),soybean trypsin (28,300).

[0023]FIG. 3 depicts HPLC chromatograms of nNOS-II reactions with BK orCit¹-BK. Sensitivities of the regions at 20-30 minutes are expanded3-fold. FIG. 3A is a chromatogram of the enzymatic reaction in which BKis the substrate. FIG. 3B is a chromatogram of the enzymatic reaction inwhich Cit¹-BK is the substrate.

[0024]FIG. 4 depicts double reciprocal plots of the inhibition of NOS-IIby [N-adamantaneacetyl-D-Arg⁰,Hyp³,Thi^(5,8),D-Phe⁷]-BK in the presenceof BK. The inhibition experiments were carried out the same as forstandard enzyme assays under initial velocity conditions. Concentrationsof [N-adamantaneacetyl-D-Arg⁰,Hyp³,Thi^(5,8),D-Phe⁷]-BK were closedcircles, 0 μM; open circles, 0.625 μM; closed squares, 1.25 μM; opensquares, 2.5 μM; closed triangles, 5 μM; open triangles, 10 μM. Thevalues are the mean of three measurements.

[0025]FIG. 5 depicts DEAE-agarose column elution profile for NOSs andprotein (absorbance at 280 nm.) from rat cerebellum. L-arginine (O) NOSactivity (calmodulin dependent) and BK () NOS activity (calmodulinindependent).

[0026]FIG. 6 depicts an 8%, silver-stained SDS-polyacrylamide gelshowing: Lane 1, cytosol (10 μg protein); Lane 2, eluate from firstDEAE-agarose column (10 μg); Lane 3, NADPH eluate from 2′-5′-ADP-agarosecolumn (2 μg protein); Lane 4, eluate from second DEAE-agarose columncontaining purified enzyme (0.5 μg protein); Lane 5, control with samplebuffer; Lane 6, molecular weight markers (Bio-Rad) of myosin (208,000).b-galactosidase (115,000) phosphorylase B (107,000), bovine serumalbumin (79,500), ovalbumin (49,500), carbonic anhydrase (34,800),soybean trypsin (28,300).

[0027]FIG. 7 depicts HPLC chromatograms of nNOS-II reactions with BK orCit¹-BK. Sensitivies of the regions at 20-30 minutes are expanded3-fold. (A) Chromatogram of enzymatic reaction with BK as substrate. (B)Chromatogram of enzymatic reaction with Cit¹-BK as substrate.

[0028]FIG. 8 depicts the oxidation of bradykinin (BK) to Cit¹.Cit⁹-BKand nitric oxide (NO) by nNOS-II.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention is directed to a novel constitutivemammalian nitric oxide synthase (NOS) that utilizes arginine-richpeptides, oligopeptides (e.g., bradykinin), and proteins, as well asL-arginine, as substrates. The present invention was begun following anearlier discovery by the inventors of a novel bacterial NOS thatutilized a range of arginine-containing polypeptides, including BK andrelated BK analogs ranging from 6-mer to 11-mer, to produce nitricoxide. (See, Chen and Rosazza, Biochem. Biophys. Res. Commun. 203:1251(1994) and Chen and Rosazza, J. Bacteriol. 177:5122 (1995)).

[0030] Although increasingly diverse biological functions are beingattributed to NO formed by three major known types of NOS (Nathan, FASEBJ. 6:3051-3064 (1992); Marletta, J. Biol. Chem. 268:12231-12234 (1993);Knowles et al., Biochem. J. 298:249-258 (1994); Griffith et al., Annu.Rev. Physiol. 57:707-736 (1995)), there are no known published reportsof arginine-containing oligopeptides, polypeptides or proteins acting asa substrate for any NOS enzyme. To the contrary, throughout the priorart, L-arginine is currently recognized as the only physiologicallyrelevant NOS-substrate.

[0031] The present inventors found that upon purification of crudesupernatant preparations from rat cerebellum using weak anion exchangechromatography (DEAE-agarose), the elution profile reproducibly producestwo calmodulin-dependent neuronal nitric oxide synthase (nNOS) peakswith L-arginine as substrate. Both peaks display the characteristiccalmodulin-dependent nNOS activity using L-arginine as substrate, asdescribed by Bredt et al., PNAS 87:682(1990). However, the novel enzymeof the second peak (fractions 15-21), which has not been studied in theprior art, further displays unique calmodulin-independent nNOS activityin the presence of an arginine-rich polypeptide substrate, such as BK.(NOS is measured by the conversion of oxyhemoglobin to methemoglobin.)This novel nitric oxide synthase has been purified to afford a6,360-fold purified enzyme preparation, and designated nNOS-II. When aspecies of nNOS-II acts upon a particular substrate, such as BK, theenzyme name may further denominate the substrate, e.g., nNOS_(BK). Itshould be emphasized, however, that BK is only one of many peptide,oligopeptide or protein substrates for nNOS-II.

[0032] Purified nNOS-II has a Mr of 105 kD by SDS-PAGE analysis and anapparent native M_(r) of 230 kD by gel filtration, indicating that theenzyme is a homodimeric protein. By comparison, the previouslydescribed, purified nNOS enzyme migrates as a single 160 kD band onSDS-PAGE, and the native enzyme appears to be a monomer. However, bothenzymes require the presence of NAPDH, FAD, FMN, Ca²⁺, andtetrahydrobiopterin cofactors for substrate oxidation to occur.

[0033] When calmodulin is present with the necessary cofactors, nNOS-IIalso oxidizes L-arginine, but at a K_(M) of 10.6 μM, slightly higherthan the previously reported K_(M) of 2 μM for nNOS. (K_(M) is thesubstrate concentration that allows the reaction to proceed at one-halfits maximum rate.) Moreover, with an L-arginine substrate, the rate ofreaction, V_(max), value displayed by nNOS-II is 0.85 μmol/min/mgprotein, while that reported for the nNOS of the prior art is 0.96μmol/min/mg protein. (V_(max) refers to the rate of the enzyme reaction,depending only upon how rapidly the substrate molecule can be processed.This rate when divided by the enzyme concentration provides the turnovernumber.)

[0034] Finally, numerous reports describe the abolishment of nNOSactivity (as defined by Bredt and Snyder) by the addition of quantitiesof L-arginine analog inhibitors. However, in marked contrast, withnNOS-II using BK as substrate, N^(G)-methyl-L-arginine(L-NMA) andN^(G)-nitro-L-arginine(L-NNA) were found to be reversible competitiveinhibitors with apparent Ki values of 8.6 μM and 23.8 μM, respectively.These values are significantly different for the reported Ki values forL-NMA of 1.4 μM and for L-NNA of 4.4 μM for nNOS with L-arginine assubstrate.

[0035] Thus, the present invention comprises a novel isoform of nitricoxide synthase, herein designated as nNOS-II, which can be readilydistinguished from all previously reported NOS species, including nNOS.The broader implications of this discovery include the futureidentification of a new class of native, recombinant, or syntheticpeptides that will function as NOS inhibitors for the modulation ofcardiovascular, gastrointestinal, or bronchial activities, forcontraceptive control, for the management of opioid withdrawal orcocaine-induced toxicity, or for the prevention or treatment of certainnitric oxide-mediated pathogenic conditions, such as ischemic stroke,diabetes, systemic hypotension, multiple sclerosis, Huntington'sdisease, Parkinson's disease, Alzheimer's disease, and the like.

[0036] It will be understood by those skilled in the art that thepresent invention is not limited to the use of any specific peptide,oligopeptide or protein as the substrate for the nNOS-II althoughpreferred substrates will be arginine-rich peptides, oligopeptides orproteins. More preferrably, the tertiary structure of such a peptide,oligopeptide or protein substrate will be such that one or more argininegroups are available to the NOS enzyme. It is most preferred that thesubstrate be an oligopeptide of managable size, such as from 6 to 11amino acid residues, wherein both the α and ω amino acids are arginine.

[0037] Exemplary peptides, oligopeptides and proteins upon which nNOS-IIhave been shown to be active are set forth in Table 1: TABLE 1Arginine-Containing Peptides or Oligopeptides as Substrates fornNOS-II^(a) Relative Activity Peptide Amino Acid Sequence (%) L-arginineN/A^(b) 100^(c) Poly-arginine N/A  30 (M_(r) 5,000) BKArg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 125 (SEQ ID NO: 1) Des-Arg¹-BKPro-Pro-Gly-Phe-Ser-Pro-Phe-Arg  94 (SEQ ID NO: 2) Des-Arg⁹-BKArg-Pro-Pro-Gly-Phe-Ser-Pro-Phe 180 (SEQ ID NO: 3) BK fragment 1-7Arg-Pro-Pro-Gly-Phe-Ser-Pro  80 (SEQ ID NO: 4) BK fragment 1-5Arg-Pro-Pro-Gly-Phe (SEQ ID NO: 5)  61 BK fragment 2-7Pro-Pro-Gly-Phe-Ser-Pro (SEQ ID NO: 6)  0 [Lys¹]-BKLys-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 113 (SEQ ID NO: 7) Lys-BKLys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 116 (SEQ ID NO: 8) Ile-Ser-BKIle-Ser-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe- 110 Arg (SEQ ID NO: 9)Met-Lys-BK Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-  0 Arg (SEQ ID NO:10)

[0038] This list, however, is intended to be merely exemplary, notlimiting in scope.

[0039] A model substrate of nNOS-II is the nonapeptide bradykinin(Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) (SEQ ID NO:1). BK was selected asa convenient and available model oligopeptide substrate for displayingthe catalytic properties of nNOS-II because:

[0040] it contains positionally different N- and C-terminal arginineresidues;

[0041] its recognized physiological role in both cardiovascular andcentral nervous systems has been well characterized;

[0042] it displays physiological and pharmacological effects similar tothose ascribed to NO; and

[0043] it has been linked as an apparent mediator of NOS activity(Regoli et al., Pharmacol. Rev. 32:1-46 (1980); Bhoola, Pharmacol. Rev.44:1-58 (1992)). In addition, BK is of ideal size (containing 9 aminoacid residues) for conversion by the nNOS-II enzyme.

[0044] In a preferred embodiment, the present invention providessubstantially pure nNOS-II protein. One specific enzyme herein providedin purified form is nNOS-II. When used with regard to nNOS-II of thepresent invention, the terms “protein” and “enzyme” are usedinterchangeably, even though technically an enzyme is a specific subsetof the category “protein.” Moreover, as used herein, a protein is saidto be “highly purified” or “substantially pure” if the specific activityof the protein cannot be significantly incresed by further purification,and if the specific activity is greater than that found in whole cellextracts containing the protein.

[0045] Any eukaryotic organism can be used as a source of nNOS-II or thegenes encoding same, as long as the source organism naturally containsthe enzyme or its equivalent. As used herein, “source organism” refersto the original organism from which the amino acid or DNA sequence isderived, regardless of the organism the enzyme expressed in orultimately isolated from. For example, a human is said to be the “sourceorganism” of nNOS-II expressed by a bacterial expression system as longas the amino acid sequence is that of human nNOS-II. The most preferredsource organism is mammalian.

[0046] A variety of methodologies known in the art can be utilized toobtain the nNOS-II proteins of the present invention. In one embodiment,the enzyme is purified from tissues or cells which naturally produce it,such as rat cerebellum. One skilled in the art can readily follow knownmethods for isolating proteins in order to obtain the nNOS-II proteins.These include, but are not limited to, immunochromotography,size-exclusion chromatography, ion-exchange chromatography, affinitychromatography, HPLC, and the methods set forth by example in thepresent disclosure. One skilled in the art can readily adapt knownpurification schemes to delete certain steps or to incorporateadditional purification procedures.

[0047] In a preferred embodiment of the invention, nNOS-II may bepurified using column chromatography. Specifically, it has been foundthat greater than one-thousand-fold purification can be achieved usingan affinity chromatography column. Since NADPH is a necessary cofactorfor enzyme activity, if one employs a solid matrix containing an NADPHmoiety or an NADPH analog, such as dextran blue, or 2′,5′-ADP agarose or2′,5′-ADP sepharose, then the NOS of the present invention will bind tothe matrix. It can be eluted using a soluble form of NADPH or an analogthereof at a concentration of about 1 to about 10 mM. It is desirablethat the preparation which is applied to the affinity chromatographycolumn first be partially purified on an ion exchange column, such as,diethylaminoethyl (DEAE) agarose. Other ion exchange columns known inthe art can also be used. The NOS of the present invention binds toDEAE-agarose and can be eluted with a sodium chloride gradient. Thegreatest peak of nNOS-II activity elutes with between about 150 mM andabout 210 mM sodium chloride. A combination of these three columnchromatography processes on a cleared brain homogenate will result in ahomogeneous preparation, as can be demonstrated by silver staining of anSDS/PAGE-separated sample, or by Western blotting.

[0048] In another embodiment, the enzyme may be purified from cellswhich have been altered to express the desired protein. As used herein,a cell is said to be “altered to express a desired protein” when thecell, through genetic manipulation, is made to produce a protein whichit normally does not produce, or which the cell normally produces at lowlevels. One skilled in the art can readily adapt procedures forintroducing and expressing either genomic or cDNA sequences into eithereukaryotic or prokaryotic cells, in order to generate a cell whichproduces the desired nNOS-II protein.

[0049] The present invention further encompasses the expression of thenNOS-II proteins (or a functional derivative thereof) in eitherprokaryotic or eukaryotic cells. A “functional derivative” of asequence, either protein or nucleic acid, is a molecule that possesses abiological activity (either functional or structural) that issubstantially similar to a biological activity of the protein or nucleicacid sequence. A functional derivative of a protein may or may notcontain post-translational modifications such as covalently linkedcarbohydrate, depending on the necessity of such modifications for theperformance of a specific function. The term “functional derivative” isintended to include the “fragments,” “segments,” “variants,” “analogs,”or “chemical derivatives” of a molecule.

[0050] As used herein, a molecule is said to be a “chemical derivative”of another molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties may improve themolecule's solubility, absorption, biological half life, and the like.The moieties may alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, andthe like. Moieties capable of mediating such effects are disclosed inRemington's Pharmaceutical Sciences (1980). Procedures for coupling suchmoieties to a molecule are well known in the art.

[0051] A “variant” or “allelic or species variant” of a protein ornucleic acid is meant to refer to a molecule substantially similar instructure and biological activity to either the protein or nucleic acid.Thus, provided that two molecules possess a common activity and maysubstitute for each other, they are considered variants as that term isused herein even if the composition or secondary, tertiary, orquaternary structure of one of the molecules is not identical to thatfound in the other, or if the amino acid or nucleotide sequence is notidentical.

[0052] Preferred prokaryotic hosts include bacteria such as E. coli,Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc. Undersuch conditions, the nNOS-II will not be glycosylated. The prokaryotichost must be compatible with the replicon and control sequences in theexpression plasmid.

[0053] However, prokaryotic systems may not prove efficacious for theexpression of all proteins. While prokaryotic expression systems, e.g.,pET3c, have been used to express high molecular weight proteins, such asa biologically active (molecular weight (M_(r))˜118 kDa)FGF-1:β-galactosidase chimera (Shi et al., submitted to J. Biol. Chem.,1996), successful folding and disulfide bond formation may be difficultto accomplish in bacteria.

[0054] Nevertheless, to express nNOS-II (or a functional derivativethereof) in a prokaryotic cell, it is necessary to operably link thenNOS-II coding sequence to a functional prokaryotic promoter. Suchpromoters may be either constitutive or, more preferably, regulatable(i.e., inducible or derepressible). Examples of constitutive promotersinclude the int promoter of bacteriophage λ, the bla promoter of theβ-lactamase gene sequence of pBR322, and the CAT promoter of thechloramphenicol acetyl transferase gene sequence of pPR325, etc.Examples of inducible prokaryotic promoters include the major right andleft promoters of bacteriophage λ (P_(L) and P_(R)), the trp, recA,lacZ, lacI, and gal promoters of E. coli, the α-amylase (Ulmanen et al.,J. Bacteriol. 162:176-182 (1985)) and the ζ-28-specific promoters of B.subtilis (Gilman et al., Gene sequence 32:11-20 (1984)), the promotersof the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology ofthe Bacilli, Academic Press, Inc., NY (1982)), and Streptomycespromoters (Ward et al., Mol. Gen. Genet. 203:468-478 (1986)). See alsoreviews by Glick (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo(Biochimie 68:505-516 (1986)); and Gottesman (Ann. Rev. Genet.18:415-442 (1984)).

[0055] Proper expression in a prokaryotic cell also requires thepresence of a ribosome binding site upstream of the genesequence-encoding sequence. Such ribosome binding sites are disclosed,for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404 (1981)).

[0056] Preferred eukaryotic hosts include yeast, fungi, insect cells,mammalian cells, either in vivo or in tissue culture. Mammalian cellswhich may be useful as hosts include HeLa cells, cells of fibroblastorigin such as VERO or CHO-K1, or cells of lymphoid origin, such as thehybridoma SP2/O-AG14 or the myeloma P3×63Sg8, and their derivatives.Preferred mammalian host cells include SP2/0 and J558L, as well asneuroblastoma cell lines such as IMR 332 that may provide bettercapacities for correct post-translational processing.

[0057] For a mammalian host, several possible vector systems areavailable for the expression of nNOS-II. A wide variety oftranscriptional and translational regulatory sequences may be employed,depending upon the nature of the host. The transcriptional andtranslational regulatory signals may be derived from viral sources, suchas adenovirus, bovine papilloma virus, Simian virus, or the like, wherethe regulatory signals are associated with a particular gene sequencewhich has a high level of expression. Alternatively, promoters frommammalian expression products, such as actin, collagen, myosin, etc.,may be employed. Transcriptional initiation regulatory signals may beselected which allow for repression or activation, so that expression ofthe gene sequences can be modulated. Of interest are regulatory signalswhich are temperature-sensitive so that by varying the temperature,expression can be repressed or initiated, or are subject to chemical(such as metabolite) regulation.

[0058] Yeast expression systems can also carry out post-translationalpeptide modifications. A number of recombinant DNA strategies existwhich utilize strong promoter sequences and high copy number of plasmidswhich can be utilized for production of the desired proteins in yeast.Yeast recognizes leader sequences on cloned mammalian gene sequenceproducts and secretes peptides bearing leader sequences (i.e.,pre-peptides). Any of a series of yeast gene sequence expression systemsincorporating promoter and termination elements from the activelyexpressed gene sequences coding for glycolytic enzymes produced in largequantities when yeast are grown in mediums rich in glucose can beutilized. Known glycolytic gene sequences can also provide veryefficient transcriptional control signals. For example, the promoter andterminator signals of the phosphoglycerate kinase gene sequence can beutilized.

[0059] The more preferred host for a protein the size of nNOS-II isinsect cells, for example the Drosophila larvae. Using insect cells ashosts, the Drosophila alcohol dehydrogenase promoter can be used (see,e.g., Rubin, G. M., Science 240:1453-1459 (1988)).

[0060] The baculovirus insect cell expression system is the mostpreferred system for expressing the soluble nNOS-II construct as acarboxy-terminal triple tandem myc-epitope repeat:glutathione-S-transferase (GST) fusion protein chimera, usingconventional PCR methods (Zhan et al., J. Biol. Chem. 269:20221-20224(1994)). These include the use of recombinant circle PCR to synthesizethe soluble nNOS-II construct, the preparation and expression of therecombinant virus, AcNPV-GsJ in Sf9 cells (Summers and Smith (1988) AManual of Methods for Baculovirus Vectors and Insect Culture Procedures(Texas Experimental Station Bulletin #1555)), the use of GST affinitychromatography (Zhan et al., 1994) and reversed phase or ion exchangeHPLC to purify the recombinant protein from Sf9 cell lysates and Mycimmunoblot analysis to monitor the purification and assess the purity ofthe nNOS-II protein.

[0061] The soluble nNOS-II construct may not only prove to be valuablefor the baculovirus expression system, but also as a construct for theexpression of a secreted and soluble nNOS-II enzyme in mammalian cellsfor implantation in vivo. Moreover, baculovirus vectors can beengineered to express large amounts of nNOS-II in insect cells (Jasny,Science 238:1653 (1987); Miller et al., in Genetic Engineering (1986),Setlow et al., eds., Plenum, Vol. 8, pp. 277-297).

[0062] As discussed above, expression of nNOS-II in eukaryotic hostsrequires the use of eukaryotic regulatory regions. Such regions will, ingeneral, include a promoter region sufficient to direct the initiationof RNA synthesis. Preferred eukaryotic promoters include: the promoterof the mouse metallothionein I gene sequence (Hamer et al., J. Mol.Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight,Cell 31:355-365 (1982)); the SV40 early promoter (Benoist et al., Nature(London) 290:304-310 (1981)); the yeast gal4 gene sequence promoter(Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982);Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

[0063] As is widely known, translation of eukaryotic mRNA is initiatedat the codon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes nNOS-II (or a functional derivativethereof) does not contain any intervening codons which are capable ofencoding a methionine (i.e., AUG). The presence of such codons resultseither in a formation of a fusion protein (if the AUG codon is in thesame reading frame as the nNOS-II coding sequence) or a frame-shift aframe-shift mutation (if the AUG codon is not in the same reading frameas the nNOS-II coding sequence).

[0064] The nNOS-II coding sequence and an operably linked promoter maybe introduced into a recipient prokaryotic or eukaryotic cell either asa non-replicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule. Sincesuch molecules are incapable of autonomous replication, the expressionof the nNOS-II may occur through the transient expression of theintroduced sequence. Alternatively, permanent expression may occurthrough the integration of the introduced sequence into the hostchromosome.

[0065] In one embodiment, a vector is employed which is capable ofintegrating the desired gene sequences into the host cell chromosome.Cells which have stably integrated the introduced DNA into theirchromosomes can be selected by also introducing one or more markerswhich allow for selection of host cells which contain the expressionvector. The marker may provide for prototrophy to an auxotrophic host,biocide resistance, e.g., antibiotics, or heavy metals, such as copper,or the like. The selectable marker gene sequence can either be directlylinked to the DNA gene sequences to be expressed, or introduced into thesame cell by co-transfection. Additional elements may also be needed foroptimal synthesis of single chain binding protein mRNA. These elementsmay include splice signals, as well as transcription promoters,enhancers, and termination signals. cDNA expression vectorsincorporating such elements include those described by Okayama, H.,Molec. Cell. Biol. 3:280 (1983).

[0066] In a preferred embodiment, the introduced sequence will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Any of a wide variety of vectors maybe employed for this purpose. Factors of importance in selecting aparticular plasmid or viral vector include: the ease with whichrecipient cells that contain the vector may be recognized and selectedfrom those recipient cells which do not contain the vector; the numberof copies of the vector which are desired in a particular host; andwhether it is desirable to be able to “shuttle” the vector between hostcells of different species.

[0067] Preferred prokaryotic vectors include plasmids, such as thosecapable of replication in E. coli (such as, for example, pBR322, ColE1,pSC101, pACYC 184, πVX). Such plasmids are, for example, disclosed byManiatis et al. (In: Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1982)). Bacillus plasmidsinclude pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan(In: The Molecular Biology of the Bacilli, Academic Press, N.Y. (1982),pp. 307-329). Suitable Streptomyces plasmids include pIJ101 (Kendall etal., J. Bacteriol. 169:4177-4183 (1987)), and streptomycesbacteriophages such as φC31 (Chater et al., In: Sixth InternationalSymposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary(1986), pp. 45-54). Pseudomonas plasmids are reviewed by John et al.(Rev. Infect. Dis. 8:693-704 (1986)), and Izaki (Jpn. J. Bacteriol.33:729-742 (1978)).

[0068] Preferred eukaryotic plasmids include BPV, vaccinia, SV40,2-micron circle, etc., or their derivatives. Such plasmids are wellknown in the art (Botstein et al., Miami Wntr. Symp. 19:265-274 (1982);Broach In: The Molecular Biology of the Yeast Saccharomyces: Life Cycleand Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., p. 445-470 (1981); Broach, Cell 28:203-204 (1982); Bollon et al.,J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, In: Cell Biology: AComprehensive Treatise, Vol. 3, Gene sequence Expression, AcademicPress, NY, pp. 563-608 (1980)).

[0069] Once the vector or DNA sequence containing the construct(s) hasbeen prepared for expression, the DNA construct(s) may be introducedinto an appropriate host cell by any of a variety of suitable means:transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,etc. After the introduction of the vector, recipient cells are grown ina selective medium, which selects for the growth of vector-containingcells. Expression of the cloned gene sequence(s) results in theproduction of nNOS-II, or fragments thereof. This can take place in thetransformed cells as such, or following the induction of these cells todifferentiate (for example, by administration of bromodeoxyuracil toneuroblastoma cells or the like).

[0070] The nNOS-II proteins (or a functional derivatives thereof) of thepresent invention can be used in a variety of procedures and methods,such as for the generation of antibodies, for use in identifyingpharmaceutical compositions, for studying DNA/protein interaction, andfor examinining the mechanism of NO synthesis.

[0071] The substantially pure peptides of the present invention may alsobe administered to a mammal intravenously, intramuscularly,subcutaneously, enterally, topically or parenterally. A “substantiallypure” or “highly purified” protein, as defined previously, is a proteinpreparation that is generally lacking in other cellular components withwhich it is normally associated in vivo.

[0072] When administering peptides by injection, the administration maybe by continuous injections, or by single or multiple injections. Thepeptides are intended to be provided to a recipient mammal in a“pharmacologically or pharmaceutically acceptable form” in an amountsufficient to “therapeutically effective.” A peptide is considered to bein “pharmaceutically or pharmacologically acceptable form” if itsadministration can be tolerated by a recipient patient. An amount issaid to be “therapeutically effective” (an “effective amount”) if thedosage, route of administration, etc., of the agent are sufficient toeffect a response to nNOS-II. Thus, the present peptides may be used toinduce, increase, enhance, control, regulate or modulate the effect ofthe nNOS-II protein, or the synthesis and expression of NO.

[0073] In another embodiment of the present invention, methods forinhibiting, decreasing or preventing the activity of the nNOS-II proteincan be achieved by providing an agent capable of binding to orinhibiting the enzyme (or a functional derivative thereof). Such agentsinclude, but are not limited to: nNOS-II inhibitors and antagonists,antisense nNOS-II, the antibodies to nNOS-II (anti-nNOS-II), and thesecondary or anti-peptide peptides of the present invention. Bydecreasing the activity of nNOS-II the effect which expression of thepeptide has on NO synthesis can be modified, regulated, controlled,inhibited or prevented.

[0074] In one example of the present invention, methods are presentedfor decreasing the activity of nNOS-II (or a functional derivativethereof) by means of an anti-sense strand of cDNA to disrupt thetranslation of the nNOS-II message. Specifically, a cell is modifiedusing routine procedures such that it expresses an antisense message, amessage which is complementary to the pseudogene message. Byconstitutively or inducibly expressing the antisense RNA, thetranslation of the nNOS-II mRNA can be regulated. Such antisensetechnology has been successfully applied to regulate the expression ofpoly(ADP-ribose) polymerase (see, Ding et al., J. Biol. Chem. 267(1992)).

[0075] In the alternative, nNOS-II activity can be prevented orinhibited by binding the peptide, oligopeptide or protein substrate usedby the enzyme, thus modifying the amount of NO can be synthesized by theenzyme. Examples of such nNOS inhibitors include peptides orpetidomimetics of structures such as bradykinin B2 receptor antagoniststhat have demonstrated nNOS-II inhibition activity, or argininederivatives, such as L-NNA or L-NMA. Not all bradykinin B2 receptorantagonists have nNOS-II inhibitory activity as demonstrated in thepresent invention; however, it is within the ordinary skill of one inthe art using known techniques and the procedures herein disclosed todetermine which peptides, oligopeptides or proteins are capable ofnNOS-II inhibition.

[0076] On the other hand, methods for stimulating, increasing orenhancing the activity of the nNOS-II peptide can be achieved byproviding an agent capable of modulating the synthesis of NO by nNOS-II(or a functional derivative thereof), or by inhibiting or preventing aninhibitory signal which would diminish or stop the activity of nNOS-IIin the system. Such agents include, but are not limited to, theanti-antisense nNOS-II peptides. By enhancing the activity of nNOS-IIthe effect which the enzyme has on NO synthesis can also be modified.

[0077] In yet another embodiment, nNOS-II (or a functional derivative orvariant thereof) can be used to produce antibodies or hybridomas. Oneskilled in the art will recognize that if an antibody is desired thatwill bind to nNOS-II such an antibody would be generated as describedabove and used as an immunogen. The resulting antibodies are screenedfor the ability to bind nNOS-II.

[0078] The antibodies utilized in the above methods can be monoclonal orpolyclonal antibodies, as well fragments of these antibodies andhumanized forms. Humanized forms of the antibodies of the presentinvention may be generated using one of the procedures known in the artsuch as chimerization or CDR grafting.

[0079] In general, techniques for preparing monoclonal antibodies arewell known in the art (Campbell, “Monoclonal Antibody Technology:Laboratory Techniques in Biochemistry and Molecular Biology,” ElsevierScience Publishers, Amsterdam, The Netherlands (1984); St. Groth et al.,J. Immunol. Methods 35:1-21 (1980). For example, in one embodiment anantibody capable of binding nNOS-II is generated by immunizing an animalwith a synthetic polypeptide whose sequence is obtained from a region ofthe nNOS-II protein.

[0080] Any animal (mouse, rabbit, etc.) which is known to produceantibodies can be utilized to produce antibodies with the desiredspecificity, although because of the large size of the nNOS-II molecule,the rabbit may be preferred. Methods for immunization are well known inthe art. Such methods include subcutaneous or interperitoneal injectionof the polypeptide. One skilled in the art will recognize that theamount of polypeptide used for immunization will vary based on theanimal which is immunized, the antigenicity of the polypeptide and thesite of injection.

[0081] The polypeptide may be modified or administered in an adjuvant inorder to increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

[0082] For monoclonal antibodies, spleen cells from the immunizedanimals are removed, fused with myeloma cells, such as SP2/0-Ag14myeloma cells, and allowed to become monoclonal antibody producinghybridoma cells. A hybridoma is an immortalized cell line which iscapable of secreting a specific monoclonal antibody.

[0083] Any one of a number of methods well known in the art can be usedto identify the hybridoma cell which produces an antibody with thedesired characteristics. These include screening the hybridomas with anELISA assay, western blot analysis, or radioimmunoassay (Lutz et al.,Exp. Cell Res. 175:109-124 (1988)).

[0084] Hybridomas secreting the desired antibodies are cloned and theclass and subclass are determined using procedures known in the art(Campbell, Monoclonal Antibody Technology: Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers,Amsterdam, The Netherlands (1984)).

[0085] For polyclonal antibodies, antibody containing antisera isisolated from the immunized animal and is screened for the presence ofantibodies with the desired specificity using one of the above-describedprocedures.

[0086] Conditions for incubating an antibody with a test sample vary.Incubating conditions depend on the format employed in the assay, thedetection methods employed the nature of the test sample, and the typeand nature of the antibody used in the assay. One skilled in the artwill recognize that any one of the commonly available immunologicalassay formats (such as, radioimmunoassays, enzyme-linked immunosorbentassays, diffusion based Ouchterlony, or rocket immunofluorescent assays,or the like) can readily be adapted to employ the antibodies of thepresent invention. Examples of such assays can be found in Chard, “AnIntroduction to Radioimmunoassay and Related Techniques” ElsevierScience Publishers, Amsterdam, The Netherlands (1986); Bullock et al.,“Techniques in Immunocytochemistry,” Academic Press, Orlando, Fla. Vol.1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, “Practice and Theory ofEnzyme Immunoassays: Laboratory Techniques in Biochemistry and MolecularBiology,” Elsevier Science Publishers, Amsterdam, The Netherlands(1985).

[0087] The anti-nNOS-II antibody and nNOS-inhibitors are also effectivewhen immobilized on a solid support. Examples of such solid supportsinclude, but are not limited to, plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, and acrylic resins, such aspolyacrylamide and latex beads. Techniques for coupling antibodies tosuch solid supports are well known in the art (Weir et al., “Handbook ofExperimental Immunology” 4th Ed., Blackwell Scientific Publications,Oxford, England, Chapter 10 (1986), Jacoby et al., Meth. Enzym. 34Academic Press, N.Y. (1974).

[0088] Additionally, one or more of the antibodies used in the abovedescribed methods can be detectably labelled prior to use. Antibodiescan be detectably labelled through the use of radioisotopes, affinitylabels (such as, biotin, avidin, etc.), enzymatic labels (such as, horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (suchas, FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures foraccomplishing such labelling are well-known in the art, for example, seeSternberger et al., J. Histochem. Cytochem. 18:315 (1970), Bayer et al.,Meth. Enzym. 62:308 (1979), Engval et al., Immunol. 109:129 (1972),Goding, J. Immunol. Meth. 13:215 (1976). The labeled antibodies of thepresent invention can be used for in vitro, in vivo, and in situ assaysto identify cells or tissues which express a specific protein or enzyme.

[0089] In an embodiment of the above methods, the antibodies arelabeled, such that a signal is produced when the antibody(s) bind to thesame molecule. One such system is described in U.S. Pat. No. 4,663,278.

[0090] The antibodies or antisense peptides of the present invention maybe administered to a mammal intravenously, intramuscularly,subcutaneously, enterally, topically or parenterally. When administeringantibodies or peptides by injection, the administration may be bycontinuous injections, or by single or multiple injections.

[0091] The antibodies or antisense peptides of the present invention areintended to be provided to a recipient mammal in a “pharmaceuticallyacceptable form” in an amount sufficient to be “therapeuticallyeffective” or an “effective amount”. As above, an amount is said to betherapeutically effective (an effective amount), if the dosage, route ofadministration, etc. of the agent are sufficient to affect the responseto nNOS-II. Thus, the present antibodies may either stimulate or enhancethe activity of the nNOS-II protein, resulting in increased NO synthsis,or they may inhibit or prevent the nNOS-II conversion of the peptide,oligopeptide or protein substrate into NO. Or, secondary antibody(s) maybe designed to affect the response to the nNOS-II per se, i.e., ananti-antibody to nNOS-II. In the alternative, either an antibody or ananti-antibody may be designed to affect only the anti-sense strand ofthe molecule.

[0092] One skilled in the art can readily adapt currently availableprocedures to generate secondary antibody peptides capable of binding toa specific peptide sequence in order to generate rationally designedantipeptide peptides, for example see Hurby et al., “Application ofSynthetic Peptides: Antisense Peptides”, In Synthetic Peptides, A User'sGuide, Freeman, N.Y., pp. 289-307 (1992), and Kaspczak et al.,Biochemistry 28:9230-8 (1989). As used herein, an agent is said to be“rationally selected or designed” when the agent is chosen based on theconfiguration of the nNOS-II peptide.

[0093] To detect secondary antibodies, or in the alternative, thelabelled primary antibody, labelling reagents may include, e.g.,chromophobic, enzymatic, or antibody binding reagents which are capableof reacting with the labelled antibody. One skilled in the art willreadily recognize that the disclosed antibodies of the present inventioncan readily be incorporated into one of the established kit formatswhich are well known in the art.

[0094] An antibody is said to be in “pharmaceutically orpharmacologically acceptable form” if its administration can betolerated by a recipient patient. The antibodies of the presentinvention can be formulated according to known methods of preparingpharmaceutically useful compositions, whereby these materials, or theirfunctional derivatives, are combined with a pharmaceutically acceptablecarrier vehicle. Suitable vehicles and their formulation, inclusive ofother human proteins, e.g., human serum albumin, are described, forexample, in Remington's Pharmaceutical Sciences, 1980).

[0095] In order to form a pharmaceutically acceptable composition whichis suitable for effective administration, such compositions will containan effective amount of an antibody of the present invention togetherwith a suitable amount of carrier. Such carriers include, but are notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and a combination thereof. The carrier composition may be sterile. Theformulation should suit the mode of administration. In addition tocarriers, the antibodies of the present invention may be supplied inhumanized form.

[0096] Humanized antibodies may be produced, for example by replacing animmunogenic portion of an antibody with a corresponding, butnon-immunogenic portion (i.e., chimeric antibodies) (Robinson et al.,International Patent Publication PCT/US86/02269; Akira et al., EuropeanPatent Application 184,187; Taniguchi, European Patent Application171,496; Morrison et al., European Patent Application 173,494; Neubergeret al., PCT Application WO 86/01533; Cabilly et al., European PatentApplication 125,023; Better et al., Science 240:1041-1043 (1988); Liu etal., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu et al., J.Immunol. 139:3521-3526 (1987); Sun et al., Proc. Natl. Acad. Sci. USA84:214-218 (1987); Nishimura et al., Canc. Res. 47:999-1005 (1987); Woodet al., Nature 314:446-449 (1985)); Shaw et al., J. Natl. Cancer Inst.80:1553-1559 (1988).

[0097] The compositions of the present invention can also include minoramounts of wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation or powder. The compositioncan be formulated as a suppository with traditional binders and carrierssuch as triglycerides. Oral formulations can include standard carrierssuch as pharmaceutically acceptable mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, etc.

[0098] In a preferred embodiment of the present invention, thecompositions are formulated in accordance with routine procedures forintravenous administration to a subject. Typically, such compositionsare carried in a sterile isotonic aqueous buffer. As needed, acomposition may include a solubilizing agent and a local anesthetic.Generally, the components are supplied separately or as a mixture inunit dosage form, such as a dry lyophilized powder in a sealed containerwith an indication of active agent. Where the composition isadministered by infusion, it may be provided with an infusion containerwith a sterile pharmaceutically acceptable carrier. When the compositionis administered by injection, an ampoule of sterile water or buffer maybe included to be mixed prior to injection.

[0099] The therapeutic compositions may also be formulated in salt form.Pharmaceutically acceptable salts include those formed with free aminogroups, such as those derived from hydrochloric, phosphoric, acetic,oxalic and tartaric acids, or formed with free carboxyl groups such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides. isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0100] The dosage of the administered agent will vary depending uponsuch factors as the patient's age, weight, height, sex, general medicalcondition, previous medical history, etc. In general, it is desirable toprovide the recipient with a dosage of the antibody which is in therange of from about 1 μg/kg to 10 mg/kg (body weight of patient),although a lower or higher dosage may be administered. Suitable rangesfor intravenous administration is typically about 20-500 μg of activecompound per kilogram body weight. Effective doses may be extrapolatedfrom dose-response curves derived from in vitro and in vivo animal modeltest systems.

[0101] Since highly purified proteins are now available, X-raycrystallography and NMR-imaging techniques can be used to identify thestructure of the enzyme. Utilizing such information, computer modelingsystems are now available that allows one to “rationally design” anagent capable of binding to a defined structure (Hodgson, Biotechnology8:1245-1247 (1990)), Hodgson, Biotechnology 9:609-613 (1991)). As usedherein, an agent is said to be “rationally designed” if it is selectedbased on a computer model of nNOS-II.

[0102] In another embodiment of the present invention, methods areprovided for modulating the translation of RNA encoding nNOS-II proteinin the cell. Specifically, said method comprises introducing into a cella DNA sequence which is capable of transcribing RNA which iscomplimentary to the RNA encoding the nNOS-II protein. By introducingsuch a DNA sequence into a cell, antisense RNA will be produced whichwill hybridize and block the translation of the nNOS-II protein.Antisense cloning has been described by Rosenberg et al., Nature313:703-706 (1985), Preiss et al., Nature 313:27-32 (1985), Melton,Proc. Natl. Acad. Sci. USA 82:144-148 (1985) and Kim et al., Cell42:129-138 (1985).

[0103] Transcription of the introduced DNA will result in multiplecopies of antisense RNA which will be complimentary to the nNOS-II. Bycontrolling the level of transcription of antisense RNA, and the tissuespecificity of expression, one skilled in the art can regulate the levelof activity of nNOS-II in specific cells within a patient.

[0104] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the previouslydescribed methods and assays.

[0105] All essential publications mentioned herein are herebyincorporated by reference.

[0106] In order that those skilled in the art can more fully understandthis invention, the following examples are set forth. These examples areincluded solely for the purpose of illustration, and should not beconsidered as expressing limitations unless so set forth in the appendedclaims.

EXAMPLES

[0107] In the following examples and protocols, restriction enzymes,ligase, labels, and all commercially available reagents were utilized inaccordance with the manufacturer's recommendations. The cell andmolecular and protein purification methods utilized in this applicationare established in the art and will not be described in detail. However,standard methods and techniques for isolation, purification, labeling,and the like, as well as the preparation of standard reagents may beperformed essentially in accordance with Molecular Cloning: A LaboratoryManual, 2nd ed., edited by Sambrook, Fritsch & Maniatis, Cold SpringHarbor Laboratory, 1989, and the revised third edition thereof, or asset forth in the literature references cited and incorporated herein.Methodologic details may be readily derived from the cited publications.

Example 1 Isolation, Purification and Characterization of nNOS-II

[0108] Using the following materials and methods, substantially purenNOS-II protein was prepared and purified from crude supernatantpreparations from rat cerebellum, and characterized as containingcalmodulin-independent NOS activity capable of catalyzing the oxidationof an arginine-rich nonapeptide, BK, to produce NO.

[0109] Materials: BK, [Thi^(5,8),D-Phe⁷]-BK, and[N-adamantaneacetyl-D-Arg⁰, Hyp³, Thi^(5,8), D-Phe⁷]-BK were purchasedfrom American Peptide Co. (San Diego, Calif.). Cit¹-BK was synthesized(University of Iowa, Protein Structure Facility) by solid phasesynthesis using 2-chlorotrityl chloride resin (Barbos et al., Int. J.Peptide Protein Res. 37:513-520 (1991)). The synthetic peptide Cit¹-BKwas greater than 98% pure by HPLC, and gave m/z 1061.3 by laserdesorption mass spectrometry. (6R)-5,6,7,8-Tetrahydrobiopterin (BH₄) wasfrom Biochemical Research Inc. (Natick, Mass.). L-arginine,N^(G)-methyl-L-arginine (L-NMA), N^(G)-nitro-L-arginine (L-NNA),2′,5′-ADP-agarose and other reagents were purchased from Sigma ChemicalCo. (St Louis, Mo.).

[0110] Enzyme Purification: Ten cerebella taken from Sprague Dailey rats(male, 250-350 g) were homogenized in 50 mL of ice-cold buffer A {10 mMTris-HCl (pH 7.5) containing 1 mM DTT, 1 mM EDTA}. All subsequentpurification procedures were carried out at 4° C. The homogenate wascentrifuged at 100,000×g for 120 min, and the supernatant was loadedonto a 25 mL DEAE-agarose column equilibrated with buffer A. The columnwas washed with 50 mL buffer A and eluted with a 200 mL linear gradientof 0-500 mM NaCl in buffer A. Fractions (4 mL) were collected andassayed for enzyme activity.

[0111] Elution profiles similar to those observed by Bredt et al. (Proc.Natl Acad. Sci. USA 87:682-685 (1990)) reproducibly gave twocalmodulin-dependent NOS activity peaks with L-arginine as substrate(see FIG. 1, fractions 8-21 (◯). However, only one of these (FIG. 1,fractions 15-21 ()) displayed calmodulin-independent NOS activity withBK as substrate. Protein absorbance at 280 nm (FIG. 1, □) from ratcerebellum was used to monitor the eluting process.

[0112] Fractions representing the active peak for BK from theDEAE-agarose column were pooled and concentrated to 15 mL by membraneultrafiltration, and loaded onto a 5 mL, 2′,5′-ADP-agarose columnequilibrated with buffer B {10 mM Tris-HCl (pH 7.0) containing 1 mM DTT,1 mM EDNA, and 10% glycerol}. The column was subsequently washed with 20mL of buffer B, 10 mL of 0.5 M NaCl in buffer B, 10 mL of 0.5 mM NADH inbuffer B, 10 mL of 0.5 mM NADP⁺ in buffer B, and 20 mL of buffer B. TheNOS activity was finally eluted with 10 mL of 10 mM NADPH in buffer B.

[0113] The affinity eluate was concentrated to 1.5 mL and loaded onto a5 mL DEAE-agarose column equilibrated with buffer B. The column waswashed with 20 mL buffer B and eluted with a 100 mL linear gradient of0-300 mM NaCl in buffer B. Fractions (2 mL) were collected and assayedfor enzyme activity, and active fractions (8 mL) were pooled andconcentrated to 0.2-0.5 mL with an Amicon PM-30 membrane to afford a6,360-fold purified enzyme preparation. The thus-purified, novelconstitutive neuronal NOS was designated as nNOS-II to differentiate itfrom the NOS isoform (nNOS) previously described by Bredt et al., supra(1990).

[0114] The purification of nNOS-II is summarized in Table 2: TABLE 2Purification of nNOS-II from rat cerebellum.* Specific Activity TotalProtein Total Activity (nmol/min/mg) Purification Step (μg) (nmol/min)protein) Recovery (%) Factor (fold) Cytosol 356,640 44.18 0.19 100 1DEAE- 69,200 27.20 0.39 61.6 2 agarose ADP-agarose 66 19.10 289.1 43.21,522 DEAE- 4 4.64 1,208.3 10.5 6,360 agarose

[0115] NOS Activity Assay: NOS activity was determinedspectrophotometrically by the rapid and quantitative oxidation ofoxyhemoglobin to methemoglobin (Feelish et al., Eur. J. Pharmacol.139:19-30 (1987); Olken et al., Biochem. Biophys. Res. Commun.177:828-833 (1991)).

[0116] Like previously characterized nNOS from cerebellum, nNOS-IIrequires NADPH, FAD, FMN, Ca^(2+, and BH) ₄ with BK as substrate. Thus,reaction mixtures for NOS assays containing 50 mM Tris-HCl buffer (pH7.5), were optimized to contain 4 μM oxyhemoglobin, 100 μM NADPH, 1 mMCaCl₂, 10 μM FAD, 10 μM FMN, 20 μM BH₄, 150 μM DTT, 100 μM BK orL-arginine and 0.1-3 μg enzyme in a final volume of 0.5 mL.

[0117] nNOS-II has stabilities similar to nNOS. In addition, nNOS-IIalso oxidizes L-arginine, but only when calmodulin is added along withthe other cofactors. Thus, calmodulin (10 μg/mL) was added to thereaction mixtures when L-arginine was used as substrate.

[0118] Determinations of Molecular Weight: The molecular mass of thedenatured purified enzyme was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to beapproximately 105 kD (see, FIG. 2). The apparent native molecular massof the enzyme was approximately 230 kD by gel filtration, indicatingthat nNOS-II is a homodimeric protein.

[0119] The native molecular weight of the purified enzyme was estimatedby analytical gel filtration chromatography carried out using an AlltechMacrosphere 150 column (7 μm, 4.6×25 cm) and a mobile phase of 10 mMTris-HCl buffer (pH 7.5) containing 1 mM DTT and 0.2 M NaCl, which wasused to equilibrate the column and to elute protein samples at a flowrate of 0.5 mL/min. Eluting protein peaks (retention volumes, R_(v))were monitored at 280 nm. Standard proteins (Mr) used (as shown in FIG.2) were bovine thyroglobulin (669,000, R_(v) 1.83 mL), horse spleenapoferritin (443,000, R_(v) 1.97 mL), sweet potato β-amylase (200,000,R_(v) 2.28 mL), yeast alcohol dehydrogenase (150,000, R_(v) 2.47 mL),and bovine serum albumin (66,000, R_(v) 2.78 mL).

[0120] The peak (R_(v) 2.25 mL) corresponding to the eluted enzyme(230,000) was collected and assayed for NOS activity.

[0121] Kinetic Determinations: Since BK contains both N- and C-terminalarginines, it was possible that BK could give rise to NO plus threedifferent nonapeptide products including: Cit¹-BK, Cit⁹-BK andCit¹,Cit⁹-BK. HPLC and amino acid sequencing were used to isolate andidentify the nonapeptide product formed when nNOS-II oxidized BK.Kinetic experiments were conducted using the standard enzyme assaydescribed above.

[0122] a) Isolation of Enzymatic Products by High Performance Liquid

[0123] Chromatography (HPLC): Enzymatic reaction mixtures contained 0.5μg purified enzyme, 50 mM Tris-HCl buffer (pH 7.5), 100 μM NADPH, 20 μMFAD, 20 μM FMN, 1 mM CaCl₂, 50 μM tetrahydrobiopterin, and 100 μMsubstrate in final volumes of 0.5 mL. Reaction mixtures were incubatedfor 120 min at 37° C., and transferred to microconcentrators (Mr cut-off10,000, Bio-Rad) and centrifuged to remove the enzyme. Fractions throughthe concentrator membrane were subjected to HPLC analyses. HPLC wasperformed with a Shimadzu LC-10AD pump and a SPD-M6A photodiode arrayUV-Vis detector set at 214-219 nm. Samples of 15 μL were resolved on aμBondapak C18 column (Waters; 10 mm; 3.9×300 mm, inside diameter)preceded by a guard column (3.9×20 mm) at a flow rate of 1 mL/min. Themobile phase consisted of mixtures of: A. 0.1% trifluoroacetic acid inwater; B. 0.095% trifluoroacetic acid in acetonitrile.

[0124] Elution was achieved with the following gradients: 0-15 min,0-15% linear gradient of B; 15-35 min, 15-20% linear gradient of B; and35-40 min, 20-0% linear gradient of B. Retention times for BK, Cit¹-BK,and Cit¹,Cit⁹-BK are 22.8 min, 24.3 min, and 26.3 min, respectively.

[0125] For amino acid sequencing, reaction mixture samples of 200 μLwere injected, and peaks at 26.3 min were collected and concentrated.Incubations without substrate and without purified enzyme were used andanalyzed as controls.

[0126] b) Amino Acid Sequencing:

[0127] Amino acid sequencing was determined by automated microsequencingwith Edman degradation reactions on a 475A Sequencer (AppliedBiosystems, Inc.) in the University of Iowa, Protein Structure Facility.The enzymatic product (2 μg) from HPLC (see, FIGS. 3A and B) wassequenced in duplicate to confirm the peptide sequence.

[0128] c) Kinetic Data:

[0129] To determine K_(M) values for BK, Cit¹-BK and L-arginine, thesubstrate concentrations used were 3.125, 6.25, 12.5, 25, and 50 μM.L-NMA and L-NNA, at concentrations of 0, 3.125, 6.25, 12.5, 25, and 50μM, were used to measure Ki values in the presence of aboveconcentrations of BK. N-adamantaneacetyl-[D-Arg⁰, Hyp³, Thi^(5,8),D-Phe⁷]-BK, at concentrations of 0, 0.625, 1.25, 2.5, 5, and 10 μM, wasused to determine its Ki value in the presence of the concentrations ofBK given above. Kinetic data were calculated by fitting experimentaldata to the EZ-FIT program (Perrella, Anal Biochem. 174:437-447 (1988)).

[0130] The only peptide product obtained during the nNOS-II oxidation ofBK was Cit¹,Cit⁹-BK, where both N- and C-terminal BK-arginines wereconverted to their corresponding citrullines (FIG. 3A). The syntheticnonapeptide Cit¹-BK in which N-terminal arginine was replaced withcitrulline was also a substrate for nNOS-II, and it, too, gaveCit¹,Cit⁹-BK (FIG. 3B). Difficulties encountered in synthesizing Cit⁹-BKprecluded its use in the present investigations.

[0131] Apparent K_(M) values for BK and Cit¹-BK were 8.5 and 6.2 μM,respectively, while apparent V_(max) values for BK and Cit¹-BK were 1.2and 1.6 μmol/min/mg protein, respectively. The kinetic results suggestthat neither Cit¹-BK nor Cit⁹-BK are likely to accumulate as productsduring the course of nNOS-II oxidations of BK.

[0132] By comparison, the apparent K_(M) of nNOS-II for L-arginine was10.6 μM, slightly higher than the reported value of 2 μM for previouslydescribed nNOS (Bredt et al., Proc. Natl Acad. Sci. USA 87:682-685(1990)). With L-arginine as substrate, nNOS-II and nNOS display apparentV_(max) values of 0.85 and 0.96 μmol/min/mg protein, respectively.

[0133] With nNOS-II and BK as substrate, typical NOS inhibitors, L-NMAand L-NNA, are reversible, competitive inhibitors with apparent Kivalues of 8.6 μM and 23.8 μM, respectively. These Ki values are,however, significantly different from previously established Ki valuesfor L-NMA of 1.4 μM and L-NNA of 4.4 μM with L-arginine as substrate fornNOS (Nathan, FASEB J. 6:3051-3064 (1992); Marletta, J. Biol. Chem.268:12231-12234 (1993); Knowles et al., Biochem. J. 298:249-258 (1994);Griffith et al., Annu. Rev. Physiol. 57:707-736 (1995)).

[0134] In addition, known potent specific B2 receptor antagonists[N-adamantaneacetyl-D-Arg⁰, Hyp³, Thi^(5,8), D-Phe⁷]-BK and [Thi^(5,8),D-Phe⁷]-BK (Lammek et al., Peptides 11:1041-1043 (1990); Lammek et al.,J. Phar. Pharmacol. 43:887-888 (1988); Austin et al., J. Physiol.478:351-356 (1994)) were examined for their possible effects with regardto nNOS-II activity. While [N-adamantaneacetyl-D-Arg⁰, Hyp³, Thi^(5,8),D-Phe⁷]-BK competitively inhibited nNOS-II activity on BK, with anapparent Ki of 2.5 mM, [Thi^(5,8), D-Phe⁷]-BK had no effect on nNOS-IIactivity over a concentration range of 0.5-100 mM.

[0135] In view of these findings, it is believed that natural endogenouspeptide substrates other than BK exist for nNOS-II. It is also believedthat oligopeptide-utilizing endothelial and macrophage NOSs will befound. The broad implications of this discovery are that arginine-richpeptides of greater or lesser size than BK can serve as NOS substratesto form NO, and that natural or synthetic peptides, oligopeptides orproteins can function as NOS.

[0136] Although the present invention has been described with referenceto the presently preferred embodiments, the skilled artisan willappreciate that various modifications, substitutions, omissions andchanges may be made without departing from the spirit of the invention.Accordingly, it is intended that the scope of the present invention belimited only by the scope of the following claims, including equivalentsthereof.

Example 2 Comparison of Reaction Rates with Bradykinin Analogs

[0137] Relative reaction rates were compared with a series of BKanalogs. As expected, most of the BK analogs studied served assubstrates for nNOS-II. Table 11 shows the relative activities of thearginine-containing peptides as substrates for nNOS-II. Different thanthat for NOS_(NOC), poly-arginine only showed 30% activity for nNOS-IIcomparing to that of arginine. However, nonapeptide BK showed moreactivity than arginine; Moreover, the octapeptide, des-Arg¹-BK, in whichthe N-terminal arginine is removed had activity similar to that ofarginine. Another octapeptide, des-Arg⁹-BK, in which the C-terminalarginine is removed had activity about one-fold higher than that ofarginine. The heptapeptide BK fragment 1-7 decreased its activity by 30%while BK fragment 1-5 had about 50% activity of that for BK. When theN-terminal arginine was replaced by lysine, nonapeptide [Lys¹]-BK hadsimilar activity to that for BK, while BK with an additional N-terminallysine, decapeptide Lys-BK, had almost the same activity. However,Lys-BK with an additional methonine, the undecapeptide Met-Lys-BK,showed no activity at all. Although the length of peptides, the positionof arginine in the peptides, and the presence of certain amino acidssuch as lysine may affect the activities for nNOS-II as substrates,there is no clear relationship between the peptide length or theposition of arginine and NOS activity. These preliminary resultsindicate that BK and analogs can directly serve as substrates fornNOS-II, although structure-activity relationship could not finallyestablished based on this study. TABLE 3 Arginine-Containing Peptides asSubstrates for nNOS-II^(a) Relative Peptide Amino acid sequence activity(%) L-Arginine N/A^(b) 100^(c) Poly-arginine N/A  30 (M_(r) 5,000) BKArg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 125 Des-Arg1-BKPro-Pro-Gly-Phe-Ser-Pro-Phe-Arg  94 Des-Arg9-BKArg-Pro-Pro-Gly-Phe-Ser-Pro-Phe 180 BK fragment 1-7Arg-Pro-Pro-Gly-Phe-Ser-Pro  80 BK fragment 1-5 Arg-Pro-Pro-Gly-Phe  61BK fragment 2-7 Pro-Pro-Gly-Phe-Ser-Pro  0 [Lys1]-BKLys-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 113 Lys-BKLys-Arg-Pro-Pro-Gly-Phe-Ser-Pro- 116 Phe-Arg Ile-Ser-BKIle-Ser-Arg-Pro-Pro-Gly-Phe-Ser-Pro- 110 Phe-Arg Met-Lys-BKMet-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-  0 Phe-Arg

Example 3

[0138] The following is hereby expressly incorporated in its entirety byreference:

[0139] Chen, Yijun and Rosazza, John P. N., “Oligopeptides as Substratesand Inhibitors for a New Constitutive Nitric Oxide Synthase from RatCerebellum”, Biochemical and Biophysical Research Communications,224:303-308 (1996), Article No. 1025.

Oligopeptides as Substrates and Inhibitors for a New Constitutive NitricOxide Synthase from Rat Cerebellum Materials and Methods

[0140] Materials, BK [Thi^(5,8), D-Phe⁷]-BK, and[N-adamantaneacetyl-D-Arg⁰, Hyp³, Thi^(5,8), D-Phe⁷]-BK were purchasedfrom American Peptide Co. (San Diego, Calif.). Cit¹-BK was synthesized(University of Iowa, Protein Structure Facility) by solid phasesynthesis using 2-chlorotrityl chloride resin (9). The synthetic peptideCit¹-BK was greater than 98% pure by HPLC, and gave m/z 1061.3 by laserdesorption mass spectrometry. (6R)-5,6,7,8-Tetrahydrobiopterin (BH₄) wasfrom Biochemical Research Inc. (Natick, Mass.), L-Arginine,N^(G)-methyl-L-arginine (L-NMA), N^(G)-nitro-L-arginine (L-NNA),2′,5′-ADP-agarose and other reagents were purchased from Sigma ChemicalCo. (St. Louis, Mo.).

[0141] NOS activity assay. NOS activity was determinedspectrophotometrically by the rapid and quantitative oxidation ofoxyhemoglobin to methemoglobin (10,11). Optimized reaction mixtures forNOS assays contained 50 mM Tris-HCl buffer (pH 7.5), and were optimizedto contain 4 μM oxyhemoglobin. 100 μM NADPH. 1 mM CaCl₂, 10 μM FAD. 10μM FMN, 20 μM BH. 150 μM DTT, 100 μM BK or L-arginine and 0.1-3 μgenzyme in a final volume of 0.5 ml. Calmodulin (10 μg/ml) was added tothe reaction mixtures when L-arginine was used as substrate.

[0142] Enzyme purification. Ten cerebella taken from Sprague Dawley rats(male. 250-350 g) were homogenized in 50 ml of ice-cold buffer A [10 mMTris-HCl (pH 7.5) containing 1 nM DTT, 1 mM EDTA), and all subsequentpurification procedures were carried out at 4° C. The homogenate wascentrifuged at 100,000×g for 120 min. and the supernatant was loadedonto a 25 ml DEAE-agarose column equilibrated with buffer A. The columnwas washed with 50 ml buffer A and eluted with a 200 ml linear gradientof 0-500 mM NaCl in buffer A. Fractions (4 ml) were collected andassayed for enzyme activity. Fractions representing the active peak forBK from the DEAE-agarose column were pooled and concentrated to 15 ml bymembrane ultrafiltration, and loaded onto a 5 ml, 2′,5′-ADP-agarosecolumn equilibrated with buffer B [10 mM Tris-HCl (pH 7.0) containing 1mM DTT, 1 mM EDTA, and 10% glycerol]. The column was subsequently washedwith 20 ml of buffer B, 10 ml of 0.5 mM NADH in buffer B, 10 ml of 0.5mM NADP⁻ in buffer B, and 20 ml of buffer B. The NOS activity wasfinally eluted with 10 ml of 10 mM NADPH in buffer B. The affinityeluate was concentrated to 1.5 ml and loaded onto a 5 ml DEAE-agarosecolumn equilibrated with buffer B. The column was washed with 20 mlbuffer B and eluted with a 100 ml linear gradient of 0-300 mM NaCl inbuffer B. Fractions (2 ml) were collected and assayed for enzymeactivity, and active fractions (8 ml) were pooled and concentrated to0.2-0.5 ml with an Amicon PM-30 membrane.

[0143] Determinations of molecular weight. The molecular weight ofdenatured purified enzyme was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The nativemolecular weight of the purified enzyme was estimated by analytical gelfiltration chromatography carried out using an Alltech Macrosphere 150column (7 μm, 4.6×25 cm) and a mobile phase of 10 mM Tris-HCl buffer (pH7.5) containing 1 mM DTT and 0.2 M NaCl which was used to equilibratethe column and to elute protein samples at a flow rate of 0.5 ml/min.Eluting protein peaks (retention volumes, R_(v)) were monitored at 280nm. Standard proteins (M_(r)) used were bovine thyroglobulin (669,000,R_(v) 1.83 ml), horse spleen apoferritin (443,000, R, 1.97 ml), sweetpotato β-amylase (200,000, R_(v) 2.28 ml), yeast alcohol dehydrogenase(150,000, R_(v) 2.47 ml), and bovine serum albumin (66,000, R_(v) 2.78ml). The peak (R_(v) 2.25 ml) corresponding to the eluted enzyme(230,000) was collected and assayed for NOS activity.

[0144] Kinetic determinations. Kinetic experiments were conducted usingthe standard enzyme assay described above. To determine Km values forBK, Cit¹-BK and L-arginine, substrate concentrations used were 3.125,6.25, 12.5, 25 and 50 μM. L-NMA and L-NNA with concentrations of BK.N-adamantaneacetyl-[D-Arg⁰, Hyp³, Thi^(5,8), D-Phe⁷]-BK withconcentrations of 0, 0.625, 1.25, 2.5, 5 and 10 μM was used to determineits Ki value in the presence of the concentrations of BK given above.Kinetic data were calculated by fitting experimental data to EZ-FITprogram (12).

[0145] Isolation of enzymatic products by high performance liquidchromatography (HPLC). Enzymatic reaction mixtures contained 0.5 μgpurified enzyme, 50 mM Tris-HCl buffer (pH 7.5), 100 μM NADPH, 20 μMFAD, 20 μM FMN. 1 mM CaCl₂, 50 μM tetrahydrobiopterin, and 100 μMsubstrate in final volumes of 0.5 ml. Reaction mixtures were TABLE 1Purification of nNOS-II from Rat Cerebellum* Total Total Purificationprotein activity Specific activity Recovery factor Step (μg) (nmol/min)(nmol/min/mg protein) (%) (fold) Cytosol 356,640 44.18 0.19 110 1DEAE-agarose 69,200 27.20 0.39 61.6 2 ADP-agarose 66 19.10 289.1 43.21,522 DEAE-agarose 4 4.64 1,208.3 10.5 6,360 #protein Assay.

[0146] incubated for 120 min at 37° C., and transferred tomicroconcentrators (Mr cut-off 10,000, Bio-Rad) and centrifuged toremove the enzyme. Fractions through the concentrator membrane weresubjected to HPLC analyses. HPLC was performed with a Shimadzu LC-10ADpump and a SPD-M6A photodiode array UV-Vis detector set at 214-219 nm.Samples of 15 μl were resolved on a μBondapak C18-column (Waters: 10 μm;3.9×300 mm, inside diameter) preceded by a guard column (3.9×20 mm) at aflow rate of 1 ml/min. The mobile phase consisted of mixtures of: A.0.1% trifluoroacetic acid in water; B. 0.095% trifluoroacetic acid inacetonitrile. Elution was achieved with the following gradients: 0-15min, 0-15% linear gradient of B; 15-35 min, 15-20% linear gradient of B;and 35-40 min. 20-0% linear gradient of B. Retention times for BK,Cit¹-BK, and Cit¹.Cit⁹-BKare 22.8 min, 24.3 min, and 26.3 min.respectively. For amino acid sequencing, reaction mixture samples of 200μl were injected and peas at 26.3 min were collected and concentrated.Incubations without substrate and without purified enzyme were used andanalyzed as controls.

[0147] Amino acid sequencing. Amino acid sequencing was determined byautomated microsequencing with Edman degradation reactions on a 475ASequencer (Applied Biosystems, Inc.) in the University of Iowa. ProteinStructure Facility. The enzymatic product (2 μg) from HPLC (FIG. 3) wassequenced in duplicate to confirm the peptide sequence.

Results and Discussion

[0148] Crude supernatant preparations from rat cerebellum containedcalmodulin-independent, NOS activity capable of catalyzing the oxidationof BK to produce NO. Since the first step in purifyingarginine-utilizing neuronal nitric oxide synthase (nNOS) from ratcerebellum (13) used weak anion exchange chromatography (DEAE-agarose),this approach was taken to partially purify the BK-utilizing NOS enzyme,Elution profiles similar to those observed by Bredt and Snyder (13)reproducibly gave two calmodulin-dependent NOS activity peaks withL-arginine as substrate. Only one of these (FIG. 1, fractions 1.5-21)displayed calmodulin-independent NOS activity with BK as substrate. Thecalmodulin-independent and BK active peak was further subjected to2′,5′-ADP-agarose affinity chromatography and a second DEAE-agarosechromatographic step to afford a 6,360-fold purified enzyme preparation(Table 1). The new constitutive neuronal NOS, designated as nNOS-II todifferentiate it from previously describe nNOS (13), has, a molecularmass of 105 KD by SDS-PAGE (FIG. 2). The apparent native molecular massof the enzyme was 230 kD by gel filtration, indicating that nNOS-II is ahomodimeric protein. Like previously characterized nNOS from cerebellum,nNOS-II requires NADPH, FAD, FMN Ca²⁺, and BH₄ with BK as substrate.nNOS-II has stabilities similar to nNOS. nNOS-II also oxidizesL-arginine, but only when calmodulin is added along with the othercofactors.

[0149] Since BK contains both N- and C-terminal arginines, it waspossible that BK could give rise to NO plus three different nonapeptideproducts including: Cit¹-BK, Cit⁹-BK and Cit¹,Cit⁹-BK. HPLC and aminoacid sequencing were used to isolate and identify the nonapeptideproduct formed when nNOS-II oxidized BK. The only peptide productobtained during the nNOS-II oxidation of BK was Cit¹,Cit⁹-BK, where bothN- and C-terminal BK-arginines were converted to their correspondingcitrullines (FIG. 3A). The synthetic nonapeptide Cit¹-BK in whichN-terminal arginine was replaced with citrulline was also a substratefor nNOS-II, and it too gave Cit¹,Cit⁹-BK (FIG. 3B). Difficultiesencountered in synthesizing Cit⁹-BK precluded its use in ,these studies.Apparent K_(M) values for BK and Cit¹-BK were 8.5 and 6.2 μM.respectively while apparent V_(max) values for BK and Cit¹-BK were 1.2and 1.6 μmol/min/mg protein. respectively. The kinetic results Suggestthat neither Cit¹-BK nor Cit⁹-BK are likely to accumulate as productsduring the course of nNOS-II oxidations of BK. The apparent Km ofnNOS-II for L-arginine was 10.6 μM, slightly higher than the reportedvalue of 2 μM for previously described nNOS (13). With L-arginine assubstrate nNOS-II and nNOS display apparent V_(max) values of 0.85 and0.96 μmol/min/mg protein, respectively.

[0150] With nNOS-II and BK as substrate, typical NOS inhibitors (1-4).L-NMA and L-NNA, were reversible, competitive inhibitors with apparentKi values of 8.6 μM and 23.8 μM respectively. These Ki values aresignificantly different than previously established Ki values for L-NMAof 1.4 μM and L-NNA of 4.4 μM with L-arginine as substrate for nNOS(1-4). Known potent specific B12receptor antagonists[N-adamantaneacetyl-D-Aro⁰, Hyp³, Thi^(5,8), D-Phe⁷]-BK and (Thi^(5,8).D-Phe⁷]-BK (14-16) were examined for their possible effects vs. nNOS-IIactivity. While [N-adamantaneacetyl-D-Arg⁰, Hyp³, Thi^(5,8), D-Phe⁷]-BKcompetitively inhibited nNOS-II activity vs. BK with an apparent Ki of2.5 μM, [Thi^(5,8), D-Phe⁷]-BK had no effect nNOS-II activity over aconcentration range of 0.5-100 μM.

[0151] This study reports the discovery of a new constitutive neuronalNOS (nNOS-II) in rat cerebellum that directly oxidizes both L-arginine(calmodulin dependent) and oligopeptides (calmodulin independent) assources of NO (FIG. 4). Peptide products, and some catalytic and kineticproperties mere defined for nNOS-II. Although NO is apparently formedfrom dipeptides like Arg-Arg and Arg-Phe by crude enzyme preparationsfrom cultured endothelial and macro-phage cells (17-19), this is thefirst report describing the oxidation of oligopeptides by a mammalianNOS, and the inhibition of an NOS by peptide antagonists. Our resultssuggest that natural endogenous peptide substrates other than BK mayexist for nNOS-II, and that oligopeptide utilizing endothelial andmacrophage NOSs remain to be identified. The broad implications of thiswork are that arginine-rich peptides of greater or lesser size than BKcan serve as NOS substrates to form NO, and that natural or syntheticpeptide antagonists can function as NOS inhibitors.

ACKNOWLEDGMENTS

[0152] The authors thank Professors Robert J. Linhardt and Michael W.Duffel for their valuable comments and, and YC thanks the Center forBiocatalysis and Bioprocessing for fellowship support.

REFERENCES

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What is claimed is:
 1. A method of regulating or controlling nitricoxide production in a mammalian subject comprising administering to themammal a nitric oxide-regulating amount of an arginine-rich peptide,oligopeptide, or protein inhibitor of nitric oxide synthase.
 2. Themethod of claim 1, wherein the nitric oxide synthase is nNOS-II.
 3. Themethod of claim 1, wherein the inhibitor is N^(G)-methyl-L-arginine. 4.The method of claim 1, wherein the inhibitor is N^(G)-nitro-L-arginine.5. The method of claim 1, wherein the inhibitor is a peptide oroligopeptide.
 6. The method of claim 1, wherein the nitric oxideproduction is increased.
 7. The method of reducing the rate of nitricoxide production in a mammalian subject comprising administering to themammal a nitric oxide inhibiting amount of a peptide, oligopeptide, orprotein inhibitor of the nitric oxide synthase of claim
 1. 8. The methodof reducing the rate of nitric oxide production in a mammalian subjectcomprising administering to the mammal a nitric oxide inhibiting amountof a peptide, oligopeptide, or protein inhibitor of the nitric oxidesynthase of claim
 2. 9. The method of claim 1, wherein the nitric oxideproduction is decreased.
 10. A method of preventing or treating a nitricoxide-mediated disease or condition in a mammalian subject comprisingadministering to the subject in need of such prevention or treatment atherapeutically effective amount of a peptide, oligopeptide or proteininhibitor of nitric oxide synthase.
 11. The method of claim 10, whereinthe nitric oxide synthase is nNOS-II.
 12. The method of claim 10,wherein the nitric oxide synthase is a mammalian brain-derived nitricoxide synthase (NOS) protein purified to an activity at least6,360-fold, said protein having a denatured molecular mass as determinedby sodium dodecyl sulfate polyacrylamide gel electrophoresis underreducing conditions of about 105 kD, and a native homodimeric molecularmass as determined by gel filtration of about 230 kD), requiring FAD,FMN, Ca²⁺ and tetrahydrobiopterin cofactors for the production of nitricoxide either from L-arginine, or an analog or derivative thereof, orfrom an arginine-rich peptide, oligopeptide, or protein substrate. 13.The method of claim 10, wherein the inhibitor isN^(G)-methyl-L-arginine.
 14. The method of claim 10, wherein theinhibitor is N^(G)-nitro-L-arginine.
 15. The method of claim 10, whereinthe inhibitor is a peptide or oligopeptide.